Effects Of Green Tea Extract And Its Characteristic Components On Copper Excretion And Hepatoprotection In TX Mice

Objective:Hepatolenticular degeneration（HLD）is an autosomal recessive genetic disease mainly characterized by copper metabolism disorder.The patients with ATP7B gene mutation cause difficulty in copper excretion from the biliary tract,and excessive copper accumulation causes oxidative stress to produce a large number of free radicals,which induces oxidative damage to tissues.The liver is the direct target organ of HLD.Liver lesions induced by excessive copper are the pathological basis of HLD patients,with liver dysfunction,liver fibrosis and cirrhosis being the most common symptoms.As one of the few treatable hereditary diseases,the current drug treatment of HLD mainly focuses on copper excretion and anti-oxidation.The clinical treatment is mainly based on copper complexing agent,but it requires long-term medication and is prone to toxic and side effects such as drug-induced liver injury,which seriously affects the condition and prognosis of patients.As a natural antioxidant,green tea can scavenge free radicals to inhibit lipid peroxidation and improve liver function,and the adsorption of tea polyphenols（GTP）and L-theanine（L-TA）have better complexation with heavy metals such as copper and iron.In order to investigate the effect of green tea extract（GTE）on HLD,this paper first investigated the effect of GTE on copper metabolism,excretion and liver injury in HLD model TX mice,and further screened the copper complex active components in GTE by ion meter.Finally,its activity was verified,which provided a theoretical basis for green tea to improve the clinical symptoms of HLD.Methods:Optimize Ultra Performance Liquid Chromatography（UPLC）chromatographic conditions.The contents of L-TA,Gallic acid（GA）,Catechin（C）,Epicatechin（EC）,Gallocatechin（GC）,Epigallocatechin（EGC）,Epicatechin gallate（ECG）and Epigallocatechin gallate（EGCG）in GTE were determined,and the quality control methods were established.HE staining（Hematoxylin-eosin）was used to observe the histopathological changes of liver tissue in TX mice before and after GTE and Penicillamine（PCA）treatment.The activities of serum alkaline phosphatase（AKP）,alanine aminotransferase（ALT）,aspartate aminotransferase（AST）,liver malondialdehyde（MDA）,total glutathione（T-GSH）and superoxide dismutase（SOD）were measured.To investigate the effects of GTE and PCA on liver function and oxidative stress in TX mice,and to explore the effect of GTE and PCA on improving the clinical symptoms of HLD.And the content of copper in liver,brain,kidney,urine and feces of mice was determined by atomic absorption spectroscopy（AAS）to investigate the effect of GTE on copper metabolism and excretion in TX mice.The screening method of Cu²⁺complexation ability was constructed by ion meter method,and the complexation reaction conditions(p H value,ionic strength,Cu²⁺concentration,compound concentration)were optimized.The complexation ability of GTE,GTP,L-TA,7 polyphenol monomers and 2 chemical monomers to Cu²⁺was evaluated,and the copper complexing active components in GTE were screened.Combined with the effects of GTP and L-TA on copper content in liver,brain,kidney,urine and feces of TX mice and biochemical indexes such as ALT,AST,AKP,MDA,T-GSH and SOD in liver tissue and serum,the effects of GTE and copper complex active components GTP and L-TA to assist in the intervention of HLD were clarified.Results:The optimized UPLC chromatographic conditions were as follows:ZORBAX RRPH Eclipse Plus C18 column（1.8μm,2.1×100 mm）;0.1%formic acid water（A）-acetonitrile（B）gradient elution;flow rate:0.25 m L/min;injection volume:2μL;detection wavelength:278 nm or 199 nm;column temperature:30°C.The quantitative analysis method of 8 representative components in GTE was established.The method has good linearity,stability,precision and accuracy,and can be used for quality control of GTE.GTE can significantly improve the morphology and structure of hepatocytes in TX mice.Compared with the model group,after intervention with different doses of GTE and PCA,the serum ALT,AST,AKP activity and liver MDA level of TX mice were significantly decreased（P<0.05,P<0.01）,and the SOD activity and GSH content were significantly increased（P<0.05,P<0.01）.The AAS results showed that the copper content in liver,brain and kidney tissues of mice in the model group was significantly higher than that in the control group,and the copper excretion in urine and feces decreased.GTE promoted the metabolism and excretion of copper in mice and reduced the copper content in tissues（P<0.05,P<0.01）.GTE improved liver function and alleviated liver injury in TX mice by promoting tissue copper excretion and inhibiting oxidative stress.The optimal conditions for the determination of Cu²⁺by ion meter were optimized.The methodological study showed that the method had good precision,stability and repeatability,and could be used to determine the complexation ability of compounds with Cu²⁺.The results of copper ion screening showed that GTE,GTP,L-TA and each monomer had strong complexation ability to Cu²⁺（83.75%-95.44%）.In addition,compared with the control group,GTP and L-TA reduced the serum ALT,AST,AKP activity and liver MDA level of TX mice,and increased the activity of SOD and GSH（P<0.05,P<0.01）.GTP and L-TA decreased the levels of ALT,AST,AKP and MDA,and increased the activities of SOD and GSH in TX mice.AAS results showed that GTP and L-TA could complex free copper in vivo,promote the excretion of copper in liver,brain and kidney tissues of TX mice through urine and feces,and reduce copper accumulation.Conclusion:GTE promoted copper excretion in urine and feces of TX mice by complexing copper ions,and reduced copper accumulation in liver,brain and kidney tissues,while regulating liver redox balance to inhibit oxidative stress and improve excessive copper-induced oxidative damage of hepatocytes.GTP and L-TA have strong antioxidant activity and can effectively complex Cu²⁺,which may be the main active components of GTE to assist in improving HLD symptoms.